Optical detection for bio-entities

ABSTRACT

An integrated semiconductor device for manipulating and processing bio-entity samples and methods are described. The device includes a lower substrate, at least one optical signal conduit disposed on the lower substrate, at least one cap bonding pad disposed on the lower substrate, a cap configured to form a capped area, and disposed on the at least one cap bonding pad, a microfluidic channel, wherein a first side of the microfluidic channel is formed on the lower substrate and a second side of the microfluidic channel is formed on the cap, a photosensor array coupled to sensor control circuitry, and logic circuitry coupled to the fluidic control circuitry, and the sensor control circuitry.

BACKGROUND

Medical technology industries, including device manufactures as well aspharmaceuticals and biologics manufacturers, have experiencedsignificant commercial and technological growth over the past severaldecades. Since the discovery of DNA, our understanding of itsbio-informational role in the development, operation, and interaction ofall living beings has significantly increased thanks to the developmentof DNA sequencing techniques over the years. Through improvement in DNAsequencing detection techniques, scientists and doctors have gainedgreater insight on diseases as well as more effective treatments forpatients based on their genetic dispositions. Thus, the use and role ofDNA sequencing results in health care has increased significantly.

DNA sequences are series of the nucleotide bases adenine, guanine,cytosine, and thymine, that dictate the formation of proteins inbiological systems. By analyzing a DNA sequence, important informationcan be gleaned for both diagnostic and therapeutic purposes.Additionally, the identification and quantification of other biologicalentities (bio-entities), such as proteins, small molecules, andpathogens has pushed forward the potential of medical knowledge tobenefit humankind.

Packaged sequencers employing electrowetting-on-dielectric (EWOD) forcontrol use amplification and labeling techniques that allow for opticaldetection by using fluorescent dyes and external optical systems withanalog-to-digital conversion systems to allow for the computerprocessing required for handling the large amounts of data produced.Many implementations of packaged EWOD sequencers have a glass substrateand a transparent electrode, which can be problematic. For example,light can be transmitted through the glass substrate and into thedroplet being analyzed, where sequencing is happening. In such case,transmission may not be efficient because of interference patterns fromdifferent transparent index of refractions as well as differentthicknesses of transparent material. In addition, the integration ofcolor filters into EWOD sequencers can reduce efficiency of light sentinto a sensor array.

Therefore, a need exists for improved bio-entity manipulation devicesand processing technologies.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a cross-sectional diagram of an EWOD apparatus.

FIG. 2 is a cross-sectional diagram of a fluidic control system thatuses electrowetting to transport and manipulate bio-entity sampledroplets.

FIG. 3 is a diagram illustrating how certain actions may be achievedusing an electrowetting fluidic control system.

FIG. 4 is a diagram of a microfluidic grid for transporting and mixingtarget bio-entity samples and biological reagents.

FIG. 5 is a cross-sectional diagram of a lower wafer for use in abio-entity manipulation and processing system according to anembodiment.

FIG. 6 is a cross-sectional diagram of a lower wafer for use in abio-entity manipulation and processing system according to anotherembodiment.

FIG. 7 is a top view diagram of a lower wafer for use in a bio-entitymanipulation and processing system according to an embodiment.

FIGS. 8A and 8B are side view diagrams illustrating optical conduits andoptical inputs on a lower wafer for use in a bio-entity manipulation andprocessing system according to an embodiment.

FIGS. 9A-9F are cross-sectional diagrams illustrating embodiments of amethod for forming a lower wafer for use in a bio-entity manipulationand processing system according to an embodiment.

FIG. 10 is a cross-sectional diagram of an upper wafer that may be usedin a bio-entity manipulation and processing system according to anembodiment.

FIGS. 11A and 11B are side view diagrams illustrating embodiments ofbonding a lower wafer and an upper wafer for use in a bio-entitymanipulation and processing system according to an embodiment.

FIG. 12 is a cross-sectional diagram of a microfluidic bio-entitymanipulation and processing system according to an embodiment.

FIG. 13 is a flowchart of a method for manipulating and processingbio-entity samples with an integrated semiconductor device.

The various features disclosed in the drawings briefly described abovewill become more apparent to one of skill in the art upon reading thedetailed description below.

DETAILED DESCRIPTION

It is to be understood that the following disclosure provides manydifferent embodiments and examples for implementing different featuresof the invention. Specific examples of components and arrangements aredescribed below to simplify the present disclosure. These are, ofcourse, merely examples and are not intended to be limiting. Moreover,the formation of a first feature over or on a second feature in thedescription that follows may include embodiments in which the first andsecond features are formed in direct contact, and may also includeembodiments in which additional features may be formed interposing thefirst and second features, such that the first and second features maynot be in direct contact. Various features in the figures may bearbitrarily drawn in different scales for the sake of simplicity andclarity. Where features depicted in the various figures are commonbetween two or more figures, the same identifying numerals have beenused for clarity of description. However, this should not be understoodas limiting such features.

FIG. 1 is a cross-sectional diagram of an electro-wetting-on-dielectric(EWOD) apparatus 100. The apparatus 100 includes a substrate 102 withthree material layers thereon. These material layers include anelectrode layer 104, a dielectric layer 106, and a hydrophobic coating108. The electrode layer 104 is coupled to a variable voltage source 110by a switch 112. Attached to the opposite end of the voltage source 110is a probe 114. As depicted in FIG. 1, the apparatus 100 positions theprobe 114 to be inserted into a droplet shown in two different states.Droplet 116A depicts the droplet in a state when no voltage is beingapplied by probe 114. Because of the hydrophobic coating 108, droplet116A has a contact angle θ₀ as shown. By applying a voltage from thevoltage source 110 through the probe 114, the contact angle can bedecreased and the contact area increased. Thus, droplet 116B is thedroplet when a voltage is applied. The contact angle is then decreasedto θ_(v), bringing the mass of the droplet 116B closer to the underlyingelectrode layer 104. The change in the contact angle caused by theapplied voltage is related to the applied voltage according to equation(1) below.

$\begin{matrix}{{{\cos\;\theta_{V}} - {\cos\;\theta_{0}}} = {\frac{{ɛɛ}_{s}}{2\gamma_{LG}t}V^{2}}} & (1)\end{matrix}$

In equation (1), V is the applied electrical potential or voltage, θ_(v)is the contact angle under applied voltage V, and θ₀ is the contactangle without applied voltage V. Other variables include: ∈, thedielectric constant of the dielectric layer 106; ∈₀, the vacuumpermittivity; γ_(LG), the surface tension; and t, the thickness ofdielectric layer 106. This manipulation of the apparent hydrophobicityof the droplet in apparatus 100 may be referred to aselectrowetting-on-dielectric (EWOD). Thus, by using EWOD, the physicalconfiguration of a droplet on a hydrophobic surface can be altered andcontrolled as seen in FIG. 1.

FIG. 2 is a cross-sectional diagram of a fluidic control system 200 thatallows for transporting and manipulating bio-entity sample dropletsusing EWOD principles. The fluidic control system 200 operates around amicrofluidic channel 202 to control a droplet 204 within the channel.Droplet 204 is a bio-entity sample droplet. A “bio-entity” or“biological entity” as used herein may refer to DNA, RNA, a protein, asmall molecule, a virus or other pathogen, or any such thing that may besequenced, identified, or quantified. Such activities may take place ina medical or industrial context. Throughout the disclosure, the exampleof DNA sequencing is presented; however, the embodiments are not limitedto this example.

As seen in FIG. 2, the bottom portion of the microfluidic channel 202 isprovided by a lower substrate 206 with several layers thereon. Theselayers include three electrodes 208A, 208B, and 208C, which aresurrounded by a first dielectric layer 210. Above the dielectric layer210 is a first hydrophobic coating 212 that provides the lower surfaceof the microfluidic channel 202.

The top surface of the microfluidic channel 202 is provided by anotherhydrophobic coating, which is formed over a upper substrate 214. Thisupper substrate 214 is a substrate upon which several material layersare deposited. These layers include a top electrode layer 216, a seconddielectric layer 218, and a second hydrophobic coating 220, which formsthe top surface of the microfluidic channel 202. The upper substrate 214is inverted and brought close to the surface of the first hydrophobiccoating 212. Thus, the droplet 204 is physically bounded by the firsthydrophobic coating 212 on the bottom and the second hydrophobic coating220 on the top.

The bottom electrodes 208A, 208B, and 208C are coupled to a switch 222capable of selecting any combination of these three electrodes. Theswitch 222, in turn is connected to a voltage source 224, the oppositeside of which is connected to the top electrode layer 216. Byselectively applying a voltage to various combinations of electrodes208A, 208B, and 208C, the electric field in which the droplet 204 islocated can be altered. In the depicted embodiment a DC potential isapplied, but in other embodiments, an AC potential may be used instead.By controlling the electric fields between the bottom electrodes 208A,208B, and 208C and the top electrode 216, the droplet 204 itself can bemanipulated and transported in various ways. This can be betterunderstood by reference to FIG. 3.

FIG. 3 is a diagram illustrating how certain actions may be achievedusing an EWOD fluidic control system. Four exemplary actions aredepicted: a lateral movement 300A, a droplet split 300B, a dropletmerger 300C, and a droplet formation 300D. These examples depict actionsperformed in the fluidic control system 200 as seen from above, lookingdown onto the droplet 204 through substrate 214.

As depicted in the lateral movement 300A, the droplet 204 is situatedabove the electrode 208B. When switch 222 is asserted so that bottomelectrode 208A is disconnected from the voltage source 224 (OFF), bottomelectrode 208B is OFF, and bottom electrode 208C is connected to thevoltage source 224 (ON), the droplet moves in the direction of electrode208C until it is located over electrode 208C.

As depicted in the droplet split 300B, droplet 204 begins situated abovebottom electrode 208B. When switch 222 is asserted so that the bottomelectrode 208B is OFF and both bottom electrodes 208A and 208C are ON,the portion of the droplet 204 that is closest to bottom electrode 208Awill move to the left and the portion of the droplet 204 that is closestto bottom electrode 208C will move to the right, causing the droplet 204to be split into a droplet 204A situated over the bottom electrode 208Cand a droplet 204B situated over the bottom electrode 208A.

As depicted in the droplet merger 300C, the droplet 204A begins situatedabove 208C and the droplet 204B begins situated over 208A. When theswitch 222 is asserted so that bottom electrodes 208A and 208C are OFFand the bottom electrode 208B is ON, the droplets 204A and 204B bothmove toward the bottom electrode 208B. The droplets 204A and 204B willmerge over the bottom electrode 208B to form a single droplet.

A droplet formation 300D is also depicted in FIG. 3. Droplet formation300D depicts the formation of a bio-entity sample droplet from a largerbio-entity sample drop. The performance of droplet formation 300D usesthe three bottom electrodes 208A, 208B, and 208C, as discussed, andfurther includes a larger electrode 302. The larger electrode 302 mayallow for the placement of a larger volume of liquid in a drop 304. Inorder to form a droplet 204, all four electrodes (302, 208A, 208B, and208C) are turned ON to pull the drop 304 out along the path indicated bythe square bottom electrodes, then bottom electrodes 208B and 208C areturned OFF. The liquid over bottom electrodes 208B and 208C is pulledaway by the ON state of the other electrodes, and pushed away by thehydrophobicity of the bottom electrodes 208B and 208C in their OFFstate. The portion of drop 304 above 208A remains to form droplet 204.

These examples assume that any other adjacent electrodes are OFF. Thelateral movement 300A, the droplet split 300B, the droplet merger 300C,and the droplet formation 300D actions may be used to manipulate andtransport droplets as they move through the microfluidic channel 202 ofFIG. 2, and also through a microfluidic grid.

FIG. 4 is a diagram of a microfluidic grid 400 for transporting andmixing target bio-entities or molecules. For example microfluidic grid400 may be used for transporting and mixing target DNA samples andbiological reagents. The microfluidic grid includes a plurality ofhorizontal and vertical paths lined by electrodes like the electrodes208A, 208B, and 208C of FIG. 2. Actions like those described inconnection with FIG. 3 may be used to move, split, merge, and formdroplets in the microfluidic grid 400.

The plurality of vertical paths is labeled as vertical paths 402A-J,while the plurality of horizontal paths is labeled as horizontal paths404A-L. Each of vertical paths 402A-J and each of horizontal paths404A-L may be formed from a plurality of linearly arranged electrodes.The spaces in between the vertical paths 402A-J and the horizontal paths404A-L may be empty space as the hydrophobic coatings 212 and 220 mayeffectively bar a droplet from “jumping” from one hydrophilic path toanother with electrodes in an ON state. In some embodiments, materialbarriers exist in the spaces between the paths.

The microfluidic grid 400 also includes a plurality of tanks from whichdroplets are introduced into the plurality of paths. Arranged along thetop are a number of reagent tanks 406A-E. In the depicted embodiment ofmicrofluidic grid 400, these reagent tanks include an adenine reagenttank 406A, a thymine reagent tank 406B, a guanine reagent tank 406C, acytosine reagent tank 406D, and a buffer tank 406E. Other embodiments ofmicrofluidic grid 400 may include other biological reagents. Dropletsmay be dispensed into the microfluidic grid 400 through vertical paths402B, 402D, 402F, 402H, and 402J, and by selectively asserting theelectrodes that make up the horizontal and vertical paths, thesedroplets may be positioned any where in the microfluidic grid 400 anddivided and mixed, or merged, with other droplets. A number of reagentdroplets, including exemplary buffer droplet 408A and exemplary adeninereagent droplet 408B, are depicted along horizontal path 404C.

Depicted on the left-hand side of microfluidic grid 400 is a number ofbio-entity sample tanks 410A-D. In the depicted embodiment, used for DNAsequences, each bio-entity sample tank contains a different target DNAfragment, labeled as D1 in target DNA fragment tank 410A, D2 in targetDNA fragment tank 410B, D3 in target DNA fragment tank 410C, and D4 intarget DNA fragment tank 410D. In embodiments used for DNA sequencingthese tanks hold fragments of a DNA sample to be sequenced. Inembodiments used for diagnosis, other types of bio-entity samples, suchas antibodies, may be present in the sample tanks.

Sequencing the entire genome of a person or pathogen in a singlesequence would require a prohibitively long amount of time. Byfragmenting a DNA sample into many samples, each sample may be processedsimultaneously in order to decrease the total time required to obtainthe entire sequence. The fragments should be labeled beforehand so thatthe individual parallel sequencing can be recombined. Each square inFIG. 4 is a target DNA fragment, such as exemplary target DNA fragment410, that can be manipulated as described above in connection with FIG.3, including being mixed with a reagent droplet for tagging. The areaunderneath the microfluidic grid 400 includes a light sensor array,which may be used to take light-based measurements in order to sequencethe target DNA fragment samples. This may be better understood withreference to FIG. 5.

FIG. 5 is a cross-sectional diagram of a lower wafer 500 having a lowersubstrate 510 for use in a microfluidic bio-entity manipulation andprocessing system. The lower substrate 510 includes a fluidic controlcircuitry area, a solid-state based photosensor area, a logic circuitryarea, and a microfluidic channel area. The circuitry and photosensorareas are formed on or in the lower substrate 510. As depicted, lowersubstrate 510 is a silicon substrate. However, in other embodiments,lower substrate 510 may be a substrate formed from another suitableelementary semiconductor, such as diamond or germanium; a suitablecompound semiconductor, such as silicon carbide, indium arsenide, orindium phosphide; or a suitable alloy semiconductor, such as silicongermanium carbide, gallium arsenic phosphide, or gallium indiumphosphide.

The fluidic control circuitry area includes fluidic control circuitry,which includes a plurality of metallization layers connected withassociated transistors and other circuit components. The sensor areaincludes a photosensor array 520 and photosensor control circuitry. Inthe depicted embodiment, the photosensor array 520 is an array oftransistor-based photosensors and is a CMOS image sensor array. However,in other embodiments the photosensor array 520 may include photodiodes,active pixel sensors, phototransistors, photoresistors, charged coupleddevices, or the like. The photosensor array 520 is controlled by thephotosensor control circuitry, which also includes a plurality oftransistors and other circuit components. Finally, in the logiccircuitry area, there is a significant amount of logic circuitry,including transistors and other circuit components. The logic circuitryallows for input to and output from the lower substrate 510. Furtherlogic circuitry is coupled to both the photosensor control circuitry andthe fluidic control circuitry, to provide both with signal processingfor optimal operation, such as analog-to-digital and digital-to-analogconversion. Fluidic control circuitry, photosensor control circuitry,and logic circuitry are embedded in an inter-level dielectric layer(ILD) 530.

On top of the ILD 530, is a plurality of bottom electrodes, much likethe bottom electrodes of FIG. 2. In FIG. 5, two bottom electrodes 540are depicted. Many more electrodes may be present in practice, but thetwo depicted are adequate for clear discussion of lower substrate 510.In the depicted embodiment, bottom electrodes 540 are made from analuminum-copper alloy. However, in other embodiments different materialsmay be used that are also suitable for electrodes. Bottom electrodes 540are solid rectangles as viewed from above. The bottom electrodes 540 arein communication with the fluidic control circuitry, and thus all may bein an ON or OFF state as described in connection with FIG. 3.

On top of and surrounding the sides of bottom electrodes 540 is adielectric layer 550. In the depicted embodiment, dielectric layer 550is a high-k dielectric layer formed by an atomic layer deposition (ALD)process, or a chemical vapor deposition (CVD) process, then followed byan annealing process. Over the dielectric layer 550 is a hydrophobiccoating 560. In the depicted embodiment, hydrophobic coating 560 is madefrom polytetrafluoroethylene (PTFE), while in other embodiments it is aself-assembled monolayer.

A portion of the dielectric layer 550 has been treated with a surfacetreatment to create a surface treated area 570. In the depictedembodiment, the surface treated area 570 may contain receptors topromote DNA sequencing, while in other embodiments, a surface treatmentwith antibody binding receptors may be applied. The surface treated area570 allows identifiable reactions to take place that give of light whena droplet containing components that react with the particular receptorsare brought into contact with the surface treated area 570. For example,a molecular tag may be added onto base pairs that combine with thetarget DNA fragment, releasing the tag upon combination, with therelease of the tag emitting a light signal.

FIG. 6 illustrates another embodiment of a lower wafer 600, that allowsthe photosensor array 620 to be closer to the surface treated area 670.In between photosensor array 620 and the surface treated area 670 is anoxide or anti-reflecting coating (ARC) layer 680. The photosensor array620 is on another substrate 690, which may be silicon. Like the lowerwafer 500, the lower wafer 600 also includes ILD 630, bottom electrodes640, dielectric layer 650, and hydrophobic coating 660.

FIG. 7 illustrates a top view of the upper wafer 500 or 600. Thedielectric layer 550 and 650 is formed on the lower substrate 510 and610 and functions as an optical signal conduit or waveguide 710 withinput structures configured to couple an input source to the opticalsignal conduit 710. Attached to the optical signal conduit 710 is awaveguide splitter 720 for splitting the optical signal conduit 710 intodifferent pathways. Although the waveguide splitter 720 is shownsplitting the optical signal conduit 710 into two pathways, it should beunderstood that more than two pathways may be formed by the waveguidesplitter 720. Also shown are electrodes 730 covered in the dielectriclayer 550 or 660 and hydrophobic coating 560 and 660, and surfacetreated area 740. Many other suitable electrode configurations may beused besides the one shown.

FIGS. 8A and 8B show the optical signal conduit along the line B-B′ inFIG. 7. FIG. 8A illustrates an optical cable 802 input. The opticalsignal conduit 804 is formed on the substrate 806. The optical cable 802may be attached to the substrate 806 so that an optical core 808 of theoptical cable 802 provides an optical path for incoming light 810 to theoptical signal conduit 804. The optical cable 802 may be attached andheld in place by an adhesive 812 such as polydimethylsiloxane (PDMS), byan adhesive fastening system, or by any other suitable attachment system

FIG. 8B is a side view diagram illustrating an alternative embodiment.The optical signal conduit 824 is formed on the substrate 826. Theoptical signal conduit 824 has a grating coupler 822. In such anembodiment, a laser or other light source may be provided remotely, andmay be directed into the grating coupler 822 where incoming light 820 itis transmitted into the optical conduit 824.

FIGS. 9A-9F are cross sectional views of a lower wafer 900 at variousstages of manufacture according to one or more embodiments. Initially,FIG. 9A illustrates a lower wafer 900 in an early stage of manufacture.An optical signal conduit 902 may be disposed on a substrate 906, withthe substrate 906 being a material such as, but not limited to, glass,silicon (Si), gallium arsenide (GaAs), fiberglass, metal, or the like.Additionally, the substrate 906 may contain circuitry such as CMOSdevices; interconnect lines; sensors; electrodes; photodetectors; dopedregions, or the like, such as photosensor arrays 520, 620; ILD 530, 630;and bottom electrodes 540, 640. In one embodiment, the optical signalconduit 902 may be patterned to disperse light, or to provide separateconduit sections. An optical signal conduit 902 may, for example, be adielectric material such as silicon nitride (Si3N4), silicon oxynitride(SiON), hafnium dioxide (HfO2), tantalum pentoxide (Ta2O5), or the like.A typical optical signal conduit 902 thickness may be between about 500angstroms and about 6000 angstroms. In one embodiment, a dry etchingtechnique may be employed to pattern the optical signal conduit 902, andmay provide better optical conduit critical dimension control than wetetching. Additionally, some embodiments may have an optical signalconduit 902 with a smooth outer surface, resulting in more efficienttransmission of an optical signal.

FIG. 9B illustrates a cross-sectional view of a lower wafer 900 afterforming a sacrificial layer 912. In one embodiment, a sacrificial layer912 may be a hard or non-polymer material such as germanium (Ge),silicon (Si), titanium tungsten alloy (TiW), aluminum (Al), or the like,and may advantageously be deposited over the substrate 906 and opticalsignal conduit 902 by plasma deposition, chemical vapor deposition,physical vapor deposition, or the like. In one embodiment, thesacrificial layer 912 may have a thickness between about 2000 angstromsand about 6000 angstroms.

FIG. 9C illustrates a cross-sectional view of a lower wafer 900 afterpatterning the sacrificial layer 912. The sacrificial layer 912 may bepatterned or removed from regions outside of the future packagingcovered area 922 via lithography, or any other suitable process, leavingsacrificial layer 912 material only in the packaging covered area 922.Removal of the sacrificial layer 912 may be accomplished by an etchantappropriate for the particular sacrificial layer 912 material,including, but not limited to, hydrogen peroxide (H2O2), phosphoric acid(H3PO4), potassium hydroxide (KOH), tetramethylammonium hydroxide(TMAH), ethylenediamine pyrocatechol (EDP), xenon diflouride (XeF2), andthe like.

FIG. 9D illustrates a cross sectional view of a lower wafer 900 afterforming a bonding layer. A bonding layer 934 may be deposited over thepatterned sacrificial layer 912 and optical signal conduit 902. In oneembodiment, the bonding layer 934 may be applied so that it lies in thebonding area 932 to cover the optical signal conduit 902 and provide apad for bonding a cap wall over the signal conduit 902. The bondinglayer 934 may be, in some embodiments, an oxide such as silicon dioxideor the like, and may be deposited via, for example, a chemical vapordeposition process, a plasma enhanced deposition process, or any othersuitable process. Alternatively, the bonding layer 934 may be a nitride,a metal layer, a polysilicon layer, or the like, and the bonding layermaterial may be selected depending on the optical signal conduit 902properties. The sacrificial layer 912 may shield the optical signalconduit 902 from an overlying bonding layer 934, in the region where thebonding layer 934 will later be removed.

In one embodiment of the present principles, it may be advantageous tohave a hard sacrificial layer 912 instead of a sacrificial photoresist(PR) under the bonding layer 934 because polymer residues couldinterfere with the surface chemistry of the lower wafer 900.Additionally, the planarization of bonding layer 934 that would bedeposited on a sacrificial photoresist layer may be problematic becausethe oxide is on a soft material: the stress and pressure fromplanarization may cause a polymer-type photoresist to deform and thebonding layer to fail during the planarization. However, a biocompatiblephotoresist may be used, and the chemistry of such a biocompatiblephotoresist may be determined by the test material intended for a cappedarea, which will be discussed later. In such an instance, abiocompatible photoresist chemistry will preferably be selected to notinterfere with the testing procedure and chemistry of any targetmolecule.

The bonding layer 934 may be deposited at a thickness over the substrate906 surface between about 4 micrometers (40,000 angstroms) and 0.5micrometers (5,000 angstroms) and may be subsequently planarized, usingfor example, a chemical mechanical polish, down to a thickness betweenabout 2 micrometers (20,000 angstroms) and about 0.4 micrometers (4,000angstroms). The bonding layer 934 may provide a planarized surfacecapable of accepting a range of bonding technologies while permitting anoptical signal conduit 902 thickness up to about 600 nanometers (6,000angstroms). Thus, one useful embodiment may be where the optical signalconduit is between about 200 nanometers (2,000 angstroms) and about 600nanometers (6,000 angstroms) thick, and the bonding layer covers theoptical signal conduit 902 while having a planarized bonding surface.

FIG. 9E illustrates a cross sectional view of a lower wafer 900 afterpatterning the bonding layer 934. The bonding layer 934 may be patternedor formed into cap bonding pads 904 by etching to remove the bondinglayer 934 material in order to define or form a packaging covered area922, with bonding layer 934 material remaining in the bonding areas 932as a target for bonding cap walls 904. In one particularly usefulembodiment, the bonding layer 934 may be etched using a dry etchtechnique, such as plasma etching or ionic sputtering. Alternatively,and depending on the bonding layer 934 material, a wet etch, or anyother type of etching, may be advantageously employed to pattern thebonding layer 934. In one embodiment, the bonding layer 934 may beplanarized prior to patterning, which may avoid damage or contaminationof portions of the substrate or optical signal conduit that may beunintentionally exposed from topography-induced insufficient mask orphotoresist coverage during patterning. Additionally, planarizing thebonding layer 934 prior to patterning reduces or prevents damage ordestruction by planarization of regions whose bonding layer has beenpatterned away.

FIG. 9F illustrates a cross-sectional view of a lower wafer 900 afterthe sacrificial layer 912 is removed, exposing the optical signalconduit 902. Removal of the sacrificial layer 912 may be performed by,for example, a wet or vapor etch in a similar manner as described abovefor the sacrificial layer 912 patterning. Thus, the optical signalconduit 902 is exposed in the packaging covered area 922.

FIG. 10 is a cross-sectional diagram of an upper wafer 1000 that may beused in a bio-entity manipulation and processing system. The upper wafer1000 includes an upper substrate 1010. In the depicted embodiment, anupper substrate 1010 is a glass or silicon wafer, and does not need tobe transparent. However, in other embodiments, upper substrate 1010 maybe a substrate formed from another suitable elementary semiconductor,such as diamond or germanium; a suitable compound semiconductor, such assilicon carbide, indium arsenide, or indium phosphide; or a suitablealloy semiconductor, such as silicon germanium carbide, gallium arsenicphosphide, or gallium indium phosphide. Over upper substrate 1010 is atop electrode 1020. In the depicted embodiment, top electrode 1020 is anindium tin oxide (ITO) layer. However, in other embodiments, topelectrode 1020 may be an aluminum layer, aluminum-copper alloy layer, oranother suitable electrode layer.

A dielectric layer 1030 is deposited over the top electrode 1020. Inthis example, the dielectric layer 1020 is a high-k dielectric layerthat has been deposited by an ALD process before being annealed.Additionally, on top of the dielectric layer 1030 is a hydrophobiccoating 1040. In the depicted embodiment, the hydrophobic coating 1040is made from PTFE, but in other embodiments the hydrophobic coating 1040is made from a self-assembling monolayer.

FIGS. 11A and 11B illustrate a bonding of a lower wafer 900 and an upperwafer 1000 for use in a bio-entity manipulation and processing system1100. FIG. 11A illustrates an embodiment of a bio-entity manipulationand processing system 1100 with cap bond pads 904 disposed under the capwall 1104 and covering the optical signal conduit 902 outside of thecapped area 1108. FIG. 11B illustrates an embodiment of a bio-entitymanipulation and processing system 1100 with cap bond pads 904 disposedin the area under the cap wall 1104 but exposing the exterior portionsof the optical signal conduit 902. The cap bond pad 904 and sacrificialmaterial 912 remaining outside the capped area 1108 may be removed toexpose the exterior portion of the optical signal conduit 902 during thesteps illustrated in FIGS. 9B through 9F. Alternatively, the exteriorportion of the optical signal conduit 902 may be exposed in a separatestep, for example, after the cap 1102 is applied to the cap bond pads904.

The cap wall 1104 may be bonded to the cap bonding pads 904 using anadhesive such as an epoxy, via fusion bonding, or any other suitabletechnique. In one useful embodiment, for example, fusion bonding withlow temperature (<300° C.) anneal may be suitable where the cap bondingpad 904 material is an oxide. The upper wafer 1000 may be bonded to thecap wall 1104 to form a cap 1102 and define the capped area 1108. Thecapped area 1108 may be provided with a gaseous environment or fluidicmaterial prior to bonding the upper wafer 1000, or via a sealableopening after the cap 1102 is bonded. The cap 1102 will preferably beconfigured to remain water- or liquid-tight in an embodiment where thecapped area maintains a fluidic material. Likewise where the capped area1108 maintains a gaseous material, the cap 1102, including the cap'sstructures and bonded seams will be gas-impermeable.

Separation of the bonding material and cap walls 1104 from the opticalsignal conduit 902 by the cap bonding pads 904 permits a planar bondingsurface, since the bonding layer 934 and cap bonding pads 904 are laidover the signal conduit 902 and substrate 906 and then planarized. Asthe bonding pad 904 is planarized, the bonding pad 904 may be used tocompensate for topography created by the optical signal conduit 902 aswell as by the substrate 906. Skilled artisans will recognize that inorder to maintain a suitable planar surface, the cap bonding pads 904will be at least as thick as the optical signal conduit 902 is high sothat the cap bonding pads 904 lie on top of the optical signal conduit902. In particularly useful embodiments, the optical signal conduit 902will be less than about 600 nanometers, with the planarized cap bondingpads 904 being thicker than the optical signal conduit 902.

FIG. 12 is a cross-sectional diagram of an integrated microfluidicbio-entity manipulation and processing system 1200 that integrates thelower wafer 500 of FIG. 5 and the upper wafer 1000 of FIG. 10. Thus FIG.12 includes the substrate 510, with the fluidic control circuitry, thephotosensor control circuitry, and the logic circuitry thereon, inaddition to the photosensor array 520 therein. An ILD 530 surroundsthose features, and the integrated lower wafer 500 includes bottomelectrodes 540 deposited thereon with an overlying dielectric layer 550.In certain regions where the dielectric layer 550 does not cover theelectrodes, the dielectric layer 550 can function as an optical signalconduit, as described with respect to FIGS. 7 through 9F. On top of thedielectric layer 550 is a hydrophobic coating 560 that serves as thebottom of a microfluidic channel 1210.

The microfluidic bio-entity manipulation and processing system 1200 alsoincludes upper wafer 1000, which includes upper substrate 1010, which inthis embodiment is a silicon substrate. Over upper substrate 1010 are atop electrode 1020, a dielectric layer 1030, and a hydrophobic coating1040. The lower wafer 500 and upper wafer 1000 are combined using themethods described with respect to FIGS. 11A and 11B so that the surfacetreated area 570 is aligned with the photosensor array 520 and so thatthe hydrophobic coatings 560 and 1040 are brought close together,without contacting, to form the microfluidic channel 1210. In thedepicted embodiment the surface treated area 570 is formed onhydrophobic coating 560, which may improve performance by bringing thesurface treated area 570 closer to photosensor array 520. The presenceof hydrophobic coating 560 below surface treated area 570, however, isnot required.

In operation, a droplet 1202 is brought into contact with the surfacetreated area 570 containing receptors using the actions depicted in FIG.3, such as the lateral movement 300A. The droplet 1202 includes a taggedbio-entity sample, such as a specific DNA base mixed in the droplet suchas the exemplary adenine reagent droplet 408B from FIG. 4. When thedroplet 1202 contacts the receptors at the surface treated area 570,chemical reactions may remove the tag from the bio-entity samples in thedroplet. The removal of the tag may enhance or intensify a photonicemission. In some embodiments, the attachment rather than the removal ofthe tag may enhance or intensify a photonic emission. The emission issensed in the photosensor array 520. This signal is captured by thephotosensor control circuitry, and transmitted to the logic circuitryfor signal processing. Depending on the frequency or color of thephotonic emission, a specific base pair may be detected. In embodiments,in which antibodies in the droplet 1202 are being tested, the emissionmay indicate the presence of the particular antibody in the bio-entitysample in droplet 1202. After the droplet 1202 has been processed inthis manner, it may be moved out of the microfluidic channel 1210, andmay be moved out of the microfluidic grid 400.

A method 1300 for manipulating and processing bio-entity samples with anintegrated semiconductor device will now be described with respect toFIG. 13. The method begins at step 1302 when a bio-entity sample dropletis obtained from a first reservoir. The first reservoir is coupled to amicrofluidic grid. The method 1300 continues to step 1304 when thebio-entity sample droplet is transported from the microfluidic grid intoa microfluidic channel using an electrowetting effect. In themicrofluidic channel, the bio-entity sample droplet contacts thereceptors on the surface treatment in the microfluidic channel. Abiochemical reaction is triggered upon contact between the bio-entitysample droplet and the receptors on the surface treatment. At step 1306,a photonic signal that is produced by the interaction of the bio-entitysample droplet and the receptors on the surface treatment is detected bya photosensor array that is formed on the lower or first substrate.

To better illustrate the method 1300 in operation, reference will bemade to the integrated microfluidic bio-entity manipulation andprocessing system 1200 of FIG. 12 and some other figures discussed abovesuch as FIG. 3 and FIG. 4. The method may also be explained withreference to other embodiments of integrated microfluidic bio-entitymanipulation and processing systems disclosed herein. Thus, reference toFIG. 12 is made by way of non-limiting example. A reservoir 410A of FIG.4 may include a larger volume of a bio-entity sample. By using theaction depicted as droplet formation 300D of FIG. 3, a bio-entity sampledroplet 1202 is formed from the larger volume and introduced into themicrofluidic grid 400 of FIG. 4. The bio-entity sample droplet 1202 istransported through microfluidic grid 400, which includes a plurality ofmicrofluidic channels, one of which is microfluidic channel 1210 of FIG.12. Microfluidic channel 1210 is located on top of a material stackdeposited on lower substrate 510, the top layer of which, hydrophobiccoating 560, supplies the bottom surface of the microfluidic channel1210. Transporting the bio-entity sample droplet 1202 through themicrofluidic channel 1210 is accomplished by using the logic circuitryto control the fluidic control circuitry.

The bio-entity sample droplet 1202 is moved through the microfluidicgrid 400 of FIG. 4 and the microfluidic channel 1210 of FIG. 12 by usingthe electrowetting effect. Bottom electrodes 540 are asserted in eitherON or OFF states as indicated by FIG. 3, in order to subject thebiological droplet 1202 to controlled hydrophobic or hydrophilicsurfaces according to the ON or OFF states of the bottom electrodes. Bycontrol of the bottom electrodes 540, and in conjunction with a topelectrode 1020, the bio-entity sample droplet 1202 is guided intocontact with the surface treated area 570, which has had a surfacetreatment applied to it. Guiding the bio-entity sample droplet 1202 intocontact with the surface treated area 570 is accomplished by having thelogic circuitry exert control over the fluidic control circuitry.

Because of the surface treatment, receptors in the surface treated area570 and the bio-entity sample droplet 1202 may undergo a biochemicalreaction which intensifies or enhances the fluorescent light signal.This light is received by a photosensor array 520. Photosensor 520detects the light and a corresponding signal is sent to the logiccircuitry for processing. The logic circuitry may interpret the signalby color or frequency to determine the biochemical reaction thatoccurred. The biochemical reaction may indicate that a specific basenucleotide was detected in a target DNA fragment, or that a particularantibody was present in the bio-entity sample droplet. After thebio-entity sample droplet 1202 has been processed, it may be removedfrom the microfluidic channel 1210. In some embodiments a bufferdroplet, such as buffer droplet 408A of FIG. 4, may be transportedthrough the microfluidic channel 1210 in order to clean it.

Additionally, in some embodiments of the method, an adenine reagentdroplet 408B obtained from the adenine reagent tank 406A in FIG. 4 iscombined with the bio-entity sample droplet 1202, using the dropletmerge 300C operation of FIG. 3. The droplet merge 300C operation may mixthe bio-entity sample droplet 1202 and the adenine reagent droplet 408Bin the microfluidic grid 400. The mixed bio-entity sample droplet 1202may then be directed into contact with the surface treated area 570 inthe microfluidic channel 1210. In other embodiments, a reagent otherthan the adenine reagent droplet 408B may be used to create a differentmixed bio-entity sample droplet 1202.

Advantages of the integrated microfluidic bio-entity manipulation andprocessing system are provided by the optical signal conduit on thesubstrate 510. Light delivery to the analysis site via the evanescentwave is done through the optical signal conduit, thus making the needfor a transparent substrate and transparent top electrode unnecessaryfor a bio-entity analysis scheme involving EWOD. This provides forgreater flexibility in the materials used. Moreover, bio-entity analysisinvolving the optical signal conduit may avoid the need for colorfilters integrated above the photosensors because the EWOD method canrestrict particular base pairs to be sequenced at the moment, avoidingthe need for color differentiation. One of the broader embodiments is anintegrated semiconductor device for manipulating and processingbio-entity samples. The device includes a lower substrate, at least oneoptical signal conduit disposed on the lower substrate, at least one capbonding pad disposed on the lower substrate and over a portion of theoptical signal conduit, a cap that includes an upper substrate andconfigured to form a capped area, and disposed on the at least one capbonding pad, a microfluidic channel, a photosensor array coupled tosensor control circuitry, and logic circuitry coupled to the fluidiccontrol circuitry and the sensor control circuitry. The at least oneoptical signal conduit extends from outside the capped area to insidethe capped area. The first side of the microfluidic channel is formed onthe lower substrate and a second side of the microfluidic channel isformed on the cap, the cap being coupled to the substrate so as toprovide the microfluidic channel for a droplet containing a bio-entitysample and the microfluidic channel being coupled to fluidic controlcircuitry. The fluidic control circuitry, the sensor control circuitry,and the logic circuitry are formed on the lower substrate.

Another of the broader embodiments is an integrated semiconductor devicefor manipulating and processing genetic samples. The device includes alower substrate, at least one optical signal conduit disposed on thelower substrate and configured to transmit light to a target molecule,at least one cap bonding pad disposed on the lower substrate and over aportion of the optical signal conduit, a cap comprising an uppersubstrate and configured to form a capped area, and disposed on the atleast one cap bonding pad, a surface treated area with receptorsdisposed within the capped area and on the lower substrate andconfigured to interact with the target molecule, a microfluidic channel,and a photodetector disposed within the lower substrate and configuredto detect a response from the target molecule. The at least one opticalsignal conduit extends from outside the capped area to inside the cappedarea. A bottom surface of the microfluidic channel is formed on thelower substrate and a top surface of the microfluidic channel is formedon the cap, the cap being coupled to the substrate so as to provide themicrofluidic channel.

Yet another of the broader embodiments is a method for manipulating andprocessing bio-entity samples with an integrated semiconductor device.The method includes providing a bio-entity sample droplet from a firstreservoir, the first reservoir coupled to a microfluidic grid,transporting the bio-entity sample droplet from the microfluidic gridinto a microfluidic channel using an electrowetting effect, thebio-entity sample droplet contacting a surface treatment in themicrofluidic channel, wherein one side of the microfluidic channel isprovided on a lower substrate, transmitting light to the surfacetreatment through an optical signal conduit disposed on the lowersubstrate, and detecting a photonic signal with a photosensor array, thephotonic signal being enhanced by an interaction of the bio-entitysample droplet and the surface treatment, the photosensor array beingformed on the lower substrate.

The preceding disclosure is submitted by way of discussion and example.It does not exhaust the full scope and spirit of the disclosure andclaims. Such variations and combinations as may be apparent to one ofskill in the art are considered to be within the scope and spirit ofthis disclosure. For instance, throughout the disclosure, DNA sequencingis presented as an example, along with antibody identification. Thescope and spirit of the disclosure extends well beyond the limitedcontext of these examples. Thus, the full extent of the disclosure islimited only by the following claims.

What is claimed is:
 1. An integrated semiconductor device formanipulating and processing bio-entity samples, the device comprising: alower substrate; at least one optical signal conduit disposed on thelower substrate; at least one cap bonding pad disposed on the lowersubstrate and over a portion of the optical signal conduit; a capcomprising an upper substrate and configured to form a capped area, anddisposed on the at least one cap bonding pad, wherein the at least oneoptical signal conduit extends from outside the capped area to insidethe capped area; a microfluidic channel, wherein a first side of themicrofluidic channel is formed on the lower substrate and a second sideof the microfluidic channel is formed on the cap, the cap being coupledto the substrate so as to provide the microfluidic channel for a dropletcontaining a bio-entity sample and the microfluidic channel beingcoupled to fluidic control circuitry; a photosensor array coupled tosensor control circuitry; and logic circuitry coupled to the fluidiccontrol circuitry and the sensor control circuitry, wherein the fluidiccontrol circuitry, the sensor control circuitry, and the logic circuitryare formed on the lower substrate.
 2. The integrated semiconductordevice of claim 1, wherein the fluidic control circuitry, the sensorcontrol circuitry, and the logic circuitry are embedded in aninter-level dielectric (ILD) layer, and further comprise a plurality ofelectrodes over the ILD layer, the plurality of electrodes being coupledto the fluidic control circuitry.
 3. The integrated semiconductor deviceof claim 1, wherein the first side of the microfluidic channelcomprises: a high-k dielectric layer; and a hydrophobic coating coveringthe high-k dielectric layer.
 4. The integrated semiconductor device ofclaim 3, wherein the hydrophobic coating is a self-assembled monolayeror a polytetrafluoroethylene layer.
 5. The integrated semiconductordevice of claim 1, wherein the second side of the microfluidic channelcomprises: a dielectric layer over the cap; and a hydrophobic coatingover the dielectric layer.
 6. The integrated semiconductor device ofclaim 1, further comprising a surface treated area, the surface treatedarea disposed on the high-k dielectric layer of the first side of themicrofluidic channel.
 7. The integrated semiconductor device of claim 1,wherein the microfluidic channel is coupled to a microfluidic grid, themicrofluidic grid being coupled to a plurality of reservoirs andconfigured to allow for transport and mixing of fluids contained in theplurality of reservoirs, the fluids including bio-entity samples andreagents.
 8. The integrated semiconductor device of claim 1, wherein theoptical signal conduit is configured to transmit light to a targetmolecule and wherein a photodetector is configured to detect a responsefrom the target molecule.
 9. The integrated semiconductor device ofclaim 1, wherein the cap is not transparent.
 10. The integratedsemiconductor device of claim 1, further comprising a plurality ofelectrodes over the cap and under a high-k dielectric layer, wherein theelectrodes are not transparent.
 11. An integrated semiconductor devicefor manipulating and processing bio-entity samples, the devicecomprising: a lower substrate; at least one optical signal conduitdisposed on the lower substrate and configured to transmit light to atarget molecule; at least one cap bonding pad disposed on the lowersubstrate and over a portion of the optical signal conduit; a capcomprising an upper substrate and configured to form a capped area, anddisposed on the at least one cap bonding pad, wherein the at least oneoptical signal conduit extends from outside the capped area to insidethe capped area; a surface treated area with receptors disposed withinthe capped area and on the lower substrate and configured to interactwith the target molecule; a microfluidic channel, wherein a bottomsurface of the microfluidic channel is formed on the lower substrate anda top surface of the microfluidic channel is formed on the cap, the capbeing coupled to the substrate so as to provide the microfluidicchannel; and a photodetector disposed within the lower substrate andconfigured to detect a response from the target molecule.
 12. Theintegrated semiconductor device of claim 11, wherein the lower substratedoes not comprise a color filter.
 13. The integrated semiconductordevice of claim 11, wherein the bottom surface and the top surface ofthe microfluidic channel comprise a hydrophobic coating.
 14. Theintegrated semiconductor device of claim 13, wherein the bottom surfaceand the top surface of the microfluidic channel further comprise ahigh-K dielectric layer.
 15. The integrated semiconductor device ofclaim 11, wherein an oxide or anti-reflective coating is disposedbetween the photodetector and the surface treated area.
 16. Theintegrated semiconductor device of claim 11, further comprising aplurality of electrodes disposed on the lower substrate.
 17. A methodfor manipulating and processing bio-entity samples with an integratedsemiconductor device, the method comprising: providing a bio-entitysample droplet from a first reservoir, the first reservoir coupled to amicrofluidic grid; transporting the bio-entity sample droplet from themicrofluidic grid into a microfluidic channel using an electrowettingeffect, the bio-entity sample droplet contacting a surface treatment inthe microfluidic channel, wherein one side of the microfluidic channelis provided on a lower substrate; transmitting light to the surfacetreatment through an optical signal conduit disposed on the lowersubstrate; and detecting a photonic signal with a photosensor array, thephotonic signal being enhanced by an interaction of the bio-entitysample droplet and the receptors on surface treatment, the photosensorarray being formed on the lower substrate.
 18. The method of claim 17,further comprising: providing a reagent droplet from a second reservoircoupled to the microfluidic grid; and mixing the bio-entity sampledroplet and the reagent droplet in the microfluidic grid to form aprepared sample droplet.
 19. The method of claim 18, whereintransporting the bio-entity sample droplet from the microfluidic gridinto a microfluidic channel comprises transporting the prepared sampledroplet into the microfluidic channel.
 20. The method of claim 17,further comprising providing an optical cable input that provides anoptical path for light to the optical signal conduit or providing agrating coupler.